The present disclosure may be applied in general to the field of chemistry, more particularly in the area of crosslinking reagents.
Methods to crosslink biomolecules such as proteins, oligonucleotides and carbohydrates to each other, to radioactive and non-radioactive metal chelates, to drugs and to surfaces have allowed development of both in vitro and in vivo diagnostic assays as well as in vivo therapies. A wide variety of methods have been developed and reviewed (Greg T. Hermanson, Bioconjugate Techniques, Academic Press).
There are a limited number of crosslinking couples, i.e., maleimide/thiol and bromoacetamide/thiol, that are routinely used to prepare conjugates for diagnostic and therapeutic uses. These reagents have limitations in that at high protein concentrations (i.e.,  greater than 5 mg/mL) protein/protein crosslinking may occur. Also, the maleimido-modified moieties have a limited half-life due to hydrolysis at neutral and basic pH. Incorporation of thiol moieties on biomolecules requires both a coupling and a subsequent activation step. The resultant thiol-modified proteins can readily oxidize to form disulfide polymerized proteins. Also macromolecules containing disulfide bonds, i.e., antibodies, are readily cleaved following activation of the thiol moiety by a reductant. Also, quantitation of the maleimido moiety is somewhat difficult and there is no means to quantify directly the level of conjugation. Therefore, it is advantageous to have a crosslinking couple that does not have these limitations.
Consequently there is a need for crosslinking couples that: bind more efficiently to surfaces; may be controlled to achieve desired crosslinking; do not lead to homobifunctional crosslinking following modification of aggregated proteins; are stable to biological conditions of varying pH and temperature; are stable in solution or when lyophilized; are one step modifications unlike those reagents currently used in the art, e.g., SATA, SPDP type reagents; can be indirectly quantified by a spectrophotometric assay; and can be used to quantify the level of conjugation by spectrophotometric means utilizing the bond formed following conjugation.
Therefore, it is an object herein to provide reagents and methods for crosslinking biomolecules to other biomolecules, polymers, metals or drugs that meet the above needs and have improved properties over known crosslinking reagents and methods.
Reagents and methods for crosslinking biomolecules to other biomolecules, polymers, metals or drugs are provided. The reagents are heterobifunctional compounds possessing, as one of the functionalities a hydrazino group, a carbonyl group, or an oxyamino group, all as defined herein. The reagents are used in the methods provided herein to afford improved crosslinking for both in vitro and in vivo diagnostic assays as well as in vivo therapies.
Provided herein are bifunctional compounds containing amine or thiol reactive moieties and a hydrazino or oxyamino moiety that may be utilized to modify small molecules, macromolecules, biomolecules and solid surfaces. A number of hydrazino moieties may be utilized including aliphatic and aromatic hydrazine derivatives, including, but not limited to, hydrazines, hydrazides, semicarbazides, carbazides, thiosemicarbazides, thiocarbazides, hydrazine carboxylates and carbonic acid hydrazines (see, e.g., FIG. 1).
In one embodiment, the reagents for use in the methods provided herein have the formula:
Bxe2x80x94Rxe2x80x94Y 
or a derivative thereof, where B is an amino or thiol reactive moiety; Y is a hydrazino group, as defined herein, an oxyamino group or a carbonyl group; and R is a divalent group having any combination of the following groups, which are combined in any order: arylene, heteroarylene, cycloalkylene, C(R10)2, xe2x80x94C(R10)xe2x95x90C(R10)xe2x80x94,  greater than Cxe2x95x90C(R12)(R13),  greater than C(R12)(R13), xe2x80x94Cxe2x89xa1Cxe2x80x94, O, S(G)a, P(J)b(R10), P(J)b(LR10), N(R10),  greater than N+(R12)(R13) and C(L); where a is 0, 1 or 2; b is 0, 1, 2 or 3; G is O or NR10; J is S or O; and L is S, O or NR10; each R10 is a monovalent group independently selected from hydrogen and M1xe2x80x94R14; each M1 is a divalent group independently having any combination of the following groups, which groups are combined in any order: a direct link, arylene, heteroarylene, cycloalkylene, C(R15)2, xe2x80x94C(R15)xe2x95x90C(R15)xe2x80x94,  greater than Cxe2x95x90C(R12)(R13),  greater than C(R12)(R13), xe2x80x94Cxe2x89xa1Cxe2x80x94, O, S(G1)a, P(J)b(R15), P(J)b(LR15), N(R15), N(COR15),  greater than N+(R12)(R13) and C(L); where a is 0, 1 or 2; b is 0, 1, 2 or 3; G1 is O or NR15; J is S or O; and L is S, O or NR15; R14 and R15 are each independently selected from the group among hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR16R17R18, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR19R20; R19 and R20 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl; R12 and R13 are selected from (i) or (ii) as follows: (i) R12 and R13 are independently selected from among hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl; or (ii) R12 and R13 together form alkylene, alkenylene or cycloalkylene; R16, R17 and R18 are each independently a monovalent group selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR19R20; and
R11, R12, R13, R14, R15, R16, R17, R18, R19 and R20 can be substituted with one or more substituents each independently selected from Z, wherein Z is selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy, S(O)hR30, NR30R31, COOR30, COR30, CONR30R31, OC(O)NR30R31, N(R30)C(O)R31, alkoxy, aryloxy, heteroaryl, heterocyclyl, heteroaryloxy, heterocyclyloxy, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, aralkoxy, heteroaralkoxy, alkoxycarbonyl, carbamoyl, thiocarbamoyl, alkoxycarbonyl, carboxyaryl, halo, pseudohalo, haloalkyl and carboxamido; h is 0, 1 or 2; and R30 and R31 are each independently selected from among hydrogen, halo, pseudohalo, cyano, azido, nitro, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy, amino, amido, alkylamino, dialkylamino, alkylarylamino, diarylamino and arylamino.
Thus, the reagents provided herein are aliphatic and aromatic crosslinking compounds that possess (i) a thiol or amine reactive group; and (ii) a hydrazino, oxyamino or carbonyl group. Thiol reactive groups are moieties that react directly with sulfhydryl groups forming stable thioether bonds. These thiol reactive groups include, but are not limted to, maleimido, xcex1-bromoacetamido and pyridyldisulfides. Amino reactive moieties are those that react directly with amine moieties forming amide bonds. These amino reactive groups include, but are not limited to, N-hydroxysuccinimidyl, p-nitrophenyl, pentafluorophenyl and N-hydroxybenzotriazolyl esters.
Hydrazino groups, as defined herein, include, but are not limited to, hydrazines, hydrazides, semicarbazides, carbazides, thiosemicarbazides, thiocarbazides, hydrazine carboxylates and carbonic acid hydrazines (see, e.g., FIG. 1). Oxyamino groups have the formula Rxe2x80x94Oxe2x80x94NH2.
In certain embodiments herein, R is an aliphatic divalent group. In these embodiments, R is a divalent group having any combination of the following groups, which are combined in any order: cycloalkylene, C(R10)2, xe2x80x94C(R10)xe2x95x90C(R10)xe2x80x94,  greater than Cxe2x95x90C(R12)(R13),  greater than C(R12)(R13), xe2x80x94Cxe2x89xa1Cxe2x80x94, O, S(G)a, P(J)b(R10), P(J)b(LR10), N(R10),  greater than N+(R12)(R13) and C(L); where the variables are as defined above.
In other embodiments herein, Y is a hydrazino group, as defined herein. In further embodiments, Y is selected from semicarbazido, thiosemicarbazido, oxyamino, carbazido or thiocarbazido. In another embodiment, R is not (CH2)n, where n is 1-12. In a further embodiment, Y is oxyamino.
Modified biomolecules are provided. These compounds are prepared by reaction of a biomolecule of interest with one of the functionalities of a bifunctional reagent, as described herein. The modified biomolecules are available for conjugation or immobilization using the remaining functional group. Biomolecules for use herein include, but are not limited to, proteins including antibodies, glycoproteins, peptides, oligonucleotides, RNA and DNA.
Conjugate vaccines are also provided. The conjugates are formed from a protein carrier, which is modified by reaction with a bifunctional reagent, as described herein. Conjugation of the resulting hydrazino or oxyamino modified protein with, e.g., a bacterial polysaccharide, that has been oxidized to produce aldehyde groups, produces a conjugate vaccine. The bifunctional reagents for use in these embodiments are, in certain embodiments, those where R is an aliphatic group.
Also provided herein are modified solid supports, including, but not limited to, synthetic polymers, beads, glass, slides, metals and particles that have been modified by reaction with a bifunctional reagent provided herein to afford modified synthetic polymers, beads, latex, glass, slides, metals, including colloidal metals, and particles that possess a hydrazino or oxyamino group. These modified solid supports are useful in immobilization of biomolecules that possess or are modified to possess a carbonyl group. The immobilized biomolecules may be used in diagnostic and therapeutic applications.